364a Monday, February 22, 2010

determine the high resolution crystal structure of the human spastin proteinboth alone and in complex with a domain of tubulin. To this end, we have ex-pressed and purified a construct of human spastin and a C-terminal tubulin con-struct, to which spastin binds. We have also crystallized spastin and collecteda 3.3 angstrom data set and are now attempting to crystallize the spastin-tubulincomplex. Visualization of such a complex will provide details as to how spastinbinds to and severs microtubules, and will also help to explain how point mu-tations in the spastin gene lead to altered protein activity and a disease pheno-type.

1880-PosCharacterization of Tau Interactions with Lipid VesiclesJocelyn M. Traina, Shana Elbaum, Elizabeth Rhoades.Yale University, New Haven, CT, USA.A number of neurodegenerative diseases, including Alzheimer’s disease, areassociated with the deposition of aggregated tau protein in the form of neuro-fibrillary tangles (NFTs). Although a causal relationship between NFTs and dis-ease has not been conclusively resolved, it is hypothesized that either the aggre-gates themselves or the process of aggregation is pathological. Tau is anintrinsically unstructured protein expressed in neurons, primarily functioningto stabilize and catalyze microtubule assembly. Studies indicate that tau bind-ing of lipid vesicles may serve as a mimic of microtubule binding. We mea-sured the affinity of the various constructs of tau encompassing the microtubulebinding region to synthetic lipid vesicles using fluorescence correlation spec-troscopy (FCS). Importantly, we observe that the binding behavior is dramati-cally affected by solution pH. At pH 7.4, the protein binds stably and we areable to extract a partition coefficient for both wild-type and the disease-associ-ated point mutant, P301L. However, at low pH, binding to lipid bilayers trig-gers rapid aggregation of the tau fragments, which we were able to confirmas amyloid using Thioflavin T binding and electron microscopy.

1881-PosTubulin is an Amphiphilic Protein Whose Interaction With Membranes isRegulated by its Charged Carboxy-Terminal TailsDan L. Sackett.NICHD / NIH, Bethesda, MD, USA.Tubulin is an acidic heterodimeric protein whose negative charges are signif-icantly (~40% of total negative charge) located on the 10-15 residue long, glu-tamic acid-rich, unstructured, carboxy-terminal tails (CTT) found on botha- and b-subunits (though not on the monomeric g-tubulin). Unsurprisingly,tubulin is a quite water-soluble protein. However, it has been consistently re-ported to be a component of highly purified membranes, including plasmamembranes, intracellular membranes such as Golgi and mitochondrial outermembranes, and vesicle membranes as in clathrin-coated endocytic vesicles.In these preparations, tubulin is non-microtubular and is tightly associatedwith the membrane, often requiring detergent for solubilization. Tubulin hasalso been shown to associate tightly with liposomes make of purified lipidonly, including neutral lipid. The exact mechanism of tubulin-membrane as-sociation has not been defined, nor has it been shown that there is only onemechanism. Tubulin could dock with lipid-embedded proteins, for example.We have shown that tubulin binds to VDAC in the mitochondrial outer mem-brane, with functional consequences for mitochondrial function, and this bind-ing requires and is mediated by the CTT. Thus the CTT can enhance mem-brane binding of tubulin. This cannot be the mechanism for liposomeinteraction, since there is no protein present other than tubulin. We showby charge-shift electrophoresis and non-ionic detergent extraction that (a) tu-bulin behaves as an amphiphilic protein, and (b) the CTT can regulate inter-action with amphipathic molecules.

Cell & Bacterial Mechanics & Motility II

1882-PosMechanics of the Cell Nucleus as a Function of Lamin Expression inGranulocyte DifferentiationAmy C. Rowat1, Diana E. Jaalouk2, David A. Weitz1, Jan Lammerding2.1Harvard University, Cambridge, MA, USA, 2Brigham Women’s Hospital/Harvard Medical School, Cambridge, MA, USA.The ability of cells to deform through narrow spaces is critical for processesranging from immune function to metastasis. Neutrophils are the most abun-dant white blood cell, which are required to transit through spaces less than1/5 of the cell’s diameter. As the nucleus is typically the stiffest organelle inthe cell, the lobulated shape of the neutrophil nucleus is thought to facilitateits transit. However, neither the mechanical properties of the nuclei, nor themechanism underlying the transition from ovoid to lobulated nuclear shape,are fully understood. We used HL60 cells as a differentiable model system

to study nuclear shape transitions and the effects on cell mechanics. To eluci-date the effects of the nuclear envelope protein, lamin A, we genetically mod-ified the cells to generate subpopulations of cells with well-defined lamin Alevels. Quantitative image analysis revealed that increased lamin A expressioninhibits the nuclear shape transition to the typical lobulated morphology. To de-termine the effect on whole-cell mechanics, we measured cell deformability byflowing cells through channels of a microfluidic device, monitoring transit timeand nuclear deformation. In addition, we performed functional assays to test ifthe impaired transition in nuclear morphology is also associated with defects inphagocytotic function, and thus reflect overall impairment of differentiationdue to increased lamin levels. These results help to elucidate the molecularmechanism of granulocyte differentiation, and may have possible implicationsfor understanding reduced immune function in aging, where lamin A has beenreported to accumulate at the nuclear envelope.

1883-PosModel for Microtubule-Actin Interactions in Growth Cone MotilityErin M. Craig1, Andrew W. Schaefer2, Paul Forscher2, Alex Mogilner1.1University of California, Davis, Davis, CA, USA, 2Yale University, NewHaven, CT, USA.A growth cone is a motile structure on the tips of axons that guides axon ex-tension to synaptic targets during nervous system development. In order totranslate chemotactic signals into a mechanical response, the microtubuleand actin filaments in the growth cone self-organize into a motile lamellipo-dial structure. A meshwork of actin filaments in the lamellipodium are contin-ually transported inward by myosin-driven forces, at a speed that matches ac-tin polymerization at the leading edge. This creates a stationary actin treadmillwhen actin adhesion to the substrate is low, and allows leading edge protru-sion when actin adhesion increases in response to guidance cues. A populationof highly dynamic microtubules that explore the P domain in stochastic burstsof growth and shrinking have also been shown to play an essential role ingrowth cone steering. Cooperation between these two filament systems isknown to be essential for directed motility. We present initial results froma theoretical model of the growth cone cytoskeleton in the lamellipodium,testing the hypothesis that dynamic microtubules and actin work cooperativelyto guide growth cone motility. We simulate dynamically unstable microtu-bules that transiently attach to actin retrograde flow, actin-myosin-adhesionforce balance, and test several scenarios for feedback between microtubulesand actin. Our theoretical work is guided by direct visualization of actinand microtubule dynamics during growth cone advance with fluorescentspeckle microscopy.

1884-PosExamining Mechanical Properties of Vertebrate Meiotic SpindlesYuta Shimamoto1, Yusuke Maeda1, Shin’ichi Ishiwata2,Albert J. Libchaber1, Tarun M. Kapoor1.1The Rockefeller University, New York, NY, USA, 2Waseda University,Tokyo, Japan.Accurate chromosome segregation during cell division relies on the self-orga-nization of a dynamic multi-component apparatus, the microtubule-based bipo-lar spindle. Through genetic, biochemical, and cell biological approaches, thecomplete ‘parts list’ of the components, including microtubule-based motorproteins, which are responsible for the assembly and maintenance of the mei-otic spindle are now available. However, how spindle self-organization is con-trolled through integration of forces generated by multiple biomolecular pro-cesses remains mysterious. Here we report a calibrated microneedle-basedsystem that allows application of sub-nanoNewton forces at specific siteswithin the metaphase spindle assembled in Xenopus egg extracts. Our set-upallows direct force measurements and can be combined with multi-modehigh resolution microscopy along with chemical perturbations of specific spin-dle components. Using this system we applied sinusoidal strain to the meta-phase spindle, keeping the range <5% to minimize nonlinearities of the re-sponse. The stress response was measured over the frequency rangedbetween 0.01 Hz and 2 Hz. Based on the resultant stress-strain relationshipwe determined the frequency-dependent mechanical properties of the spindle.The spindle response showed a typical characteristic time-scale of ~50 s, atwhich the mechanical property changed from solid-like to liquid-like. Addi-tional experiments examining stress relaxation, which may reflect the dynamicsof structural reorganization, revealed a similar time-scale. This transition disap-peared when the spindles were treated with AMPPNP, a slow-hydrolyzing ATPanalog, suggesting that the characteristic time-scale is determined by an ATP-dependent processes within the spindle. The contribution of key mitotic motorssuch as dynein and kinesin is being examined by pharmacological or immuno-logical perturbations. Together, these analyses will allow us discuss models forhow forces in the self-organizing meiotic spindle are integrated.